Cryopreservation provides a means for the stable, long-term storage of biological plant and/or animal and/or microbial cells, tissues or organs, including plant zyotic or somatic embryos and meristems at ultra-low temperatures, eg 77.degree. K. (-196.degree. C.). For example, such a technique circumvents reduced morphological competence (totipotency), genetic change and secondary product formation of plant cell cultures associated with long-term culture at normal growth temperatures.
Cryopreservation comprises many steps, of which controlled freezing is only one; successful recovery is dependent on the combined effects of cryogenic and pre- and post-freeze treatments. The transition of tissues between low and physiologically normal temperatures and oxygen tensions causes respiratory imbalances which stimulate the production of toxic oxygen radicals as described for example by Fuller, B J, Gower, J D and Green, C J in "Free radical damage and organ preservation: fact or fiction", Cryobiology," 25, 377-393 (1988).
Physiological investigations of cryopreserved cells have shown that respiratory impairment occurs primarily, but not exclusively, during early post-freeze thaw recovery as described for example by Cella R, Colombo R, Galli M G, Nielsen M G, Rollo F and Sala F in "Freeze-preservation of rice cells: a physiological study of freeze/thawed cells", Physiologia Plantarum 55, 279-284 (1982).
It is an object of the present invention to provide a method for the recovery of living cells which have been subjected to cryopreservation in which the aforementioned respiratory impairment is reduced, whereby normal cell growth and function is promoted.